Susceptibility of Gram-negative pathogens collected in Israel to ceftolozane/tazobactam, imipenem/relebactam and comparators: SMART 2018–22

Abstract Objectives To assess the in vitro antimicrobial activity of ceftolozane/tazobactam, imipenem/relebactam and comparator agents against clinical isolates of Gram-negative bacilli collected in Israel from 2018 to 2022. Methods Six clinical laboratories each collected up to 250 consecutive Gram-negative isolates per year from patients with bloodstream, intra-abdominal, lower respiratory tract and urinary tract infections. MICs were determined by CLSI broth microdilution and interpreted with 2024 EUCAST breakpoints. Acquired β-lactamase gene carriage was investigated for most ceftolozane/tazobactam- and imipenem/relebactam-resistant isolates. Results Among the full collection of Enterobacterales (n = 4420), 95.1% were susceptible to ceftolozane/tazobactam, including 95.3% of putative AmpC/ESBL-positive, non-carbapenem-resistant Enterobacterales (CRE) phenotype Escherichia coli and 86.6% of AmpC/ESBL-positive, non-CRE phenotype Klebsiella pneumoniae. Overall, 99.8% of non-Morganellaceae Enterobacterales (n = 3723) were imipenem/relebactam susceptible including 98% of the MDR isolates. Most Pseudomonas aeruginosa isolates (n = 1182) were inhibited by ceftolozane/tazobactam (93.9% susceptible) and imipenem/relebactam (94.7%). Imipenem/relebactam retained activity against ≥78% of cefepime-resistant, ceftazidime-resistant, and piperacillin/tazobactam-resistant P. aeruginosa, while ceftolozane/tazobactam inhibited the greatest percentage of meropenem-resistant P. aeruginosa (67.4%) among comparator β-lactam antimicrobials. Molecular characterization showed the majority of imipenem/relebactam-resistant Enterobacterales harboured a metallo-β-lactamase, while half of the ceftolozane/tazobactam-resistant Enterobacterales carried an acquired ESBL or AmpC. Most of the imipenem/relebactam- and ceftolozane/tazobactam-resistant P. aeruginosa characterized did not possess acquired β-lactamases. Conclusions Recent clinical isolates of Enterobacterales and P. aeruginosa collected in Israel were highly susceptible to ceftolozane/tazobactam and imipenem/relebactam.


Introduction
Ceftolozane/tazobactam and imipenem/relebactam are newer β-lactam/β-lactamase inhibitor combinations that have been approved by both the FDA and EMA for treatment of serious infections, including hospital-acquired bacterial pneumonia.Routine surveillance of these newer agents is critical in the ongoing effort to define their utility and appropriate use.Surveillance data describing in vitro susceptibility testing results for ceftolozane/tazobactam and imipenem/relebactam against clinical isolates of Gram-negative bacilli from Israel have been generally presented as a part of the Middle East region as a whole, 1-3 and data specific to isolates collected in Israel are sparse.Thus, we evaluated the activity of these two agents and relevant comparators against isolates of Gram-negative bacilli collected by clinical laboratories in Israel as part of the Study for Monitoring Antimicrobial Resistance Trends (SMART) global surveillance programme.

Bacterial isolates and antimicrobial susceptibility testing
From 2018 to 2022, six clinical laboratories in Israel participated in the SMART programme.Each laboratory collected consecutive, aerobic or facultative Gram-negative isolates from intra-abdominal, urinary tract, lower respiratory tract and bloodstream infections.Only one isolate per patient per species per year was accepted.All isolates were sent to a central laboratory (IHMA, Monthey, Switzerland), where species identity was confirmed using MALDI-TOF mass spectrometry (Bruker Daltonics, Billerica, MA, USA) and antimicrobial susceptibility performed following the CLSI reference broth microdilution method. 4MICs were interpreted using 2024 EUCAST breakpoints. 5EUCAST does not publish breakpoints for imipenem/relebactam against Morganellaceae family (Proteus, Providencia and Morganella spp.) because they display intrinsic, lowered susceptibility to imipenem by a mechanism independent of β-lactamase production 6 and relebactam does not improve the activity of imipenem against Morganellaceae.Therefore, imipenem/relebactam susceptibility was analysed for non-Morganellaceae Enterobacterales (NME) only.

Screening for β-lactamase genes
Isolates meeting the following phenotypic criteria were screened for β-lactamase genes: NME isolates (excluding Serratia spp.) testing with imipenem or imipenem/relebactam MIC values of ≥2 mg/L and Pseudomonas aeruginosa isolates testing with imipenem or imipenem/relebactam MIC values of ≥4 mg/L; Enterobacterales and P. aeruginosa isolates testing with ceftolozane/tazobactam MIC values of ≥4 and ≥8 mg/L, respectively.Published multiplex PCR assays were used to screen for β-lactamase genes as described previously. 8,9For P. aeruginosa collected in 2020-22 only, isolates were characterized by short-read whole-genome sequencing (Illumina Hiseq 2 × 150-bp reads) to a targeted coverage depth of 100× 10 and analysed using the CLC Genomics Workbench (Qiagen).The ResFinder database was used to detect β-lactamase genes. 11In total, 214 NME were molecularly characterized.Per SMART protocol for P. aeruginosa, a representative sample of approximately 75% of isolates meeting the criteria for molecular characterization was characterized for β-lactamase genes (54 randomly selected isolates of 293 that qualified were not characterized).

Results
A brief summary of the demographic and clinical characteristics associated with all isolates of Enterobacterales and P. aeruginosa collected in Israel in 2018-22 is provided in Table S1 (available as Supplementary data at JAC-AMR Online).

Discussion
Previous reports on antimicrobial resistance in Israel are limited.Often Israel is included as part of broader Middle Eastern or European data sets, making interpretation of the information specific to Israel difficult to ascertain, although, in general, resistance rates tend to be lower than other countries in the region.For example, Sader et al. 13 reported that the percentages of Israeli isolates susceptible to ceftolozane/tazobactam among Enterobacterales (93.3%) and P. aeruginosa (98.2%) collected from 2012 to 2018 were higher than that of most surrounding countries.Similarly, an earlier SMART publication showed that 94.5% (n = 1126) of P. aeruginosa collected in Israel were ceftolozane/tazobactam susceptible, the second highest percentage among countries surveyed in the Middle East/Africa region. 14Data generated by the SENTRY surveillance programme in 2013-16 noted a 22.8% MDR rate among Enterobacterales collected in Israel, 15 nearly identical to that reported here (22.6%), values slightly lower than the 30% found in a recent mini-review, 16 with the variation likely due to differences in isolate infection sources and patient populations.The current study is unique in that it is the first to report Israel-specific imipenem/relebactam susceptibility data for both Enterobacterales and P. aeruginosa, while confirming high rates of susceptibility of P. aeruginosa to ceftolozane/tazobactam.
Ceftolozane/tazobactam inhibited 95% of Enterobacterales, including 95% of the putative AmpC/ESBL-positive non-CRE phenotype E. coli and 87% of AmpC/ESBL-positive non-CRE phenotype K. pneumoniae.The activity of ceftolozane/tazobactam against P. aeruginosa (94% susceptible) surpassed that of standard first-line empiric agents (piperacillin/tazobactam, cefepime and ceftazidime) and a commonly used carbapenem, meropenem.Amikacin and colistin each inhibited >95% of the P. aeruginosa; however, despite their excellent in vitro activity, it is important to note the practical limitations associated with these agents in treating Gram-negative infections.EUCAST only publishes bracketed colistin and amikacin (systemic infections) MIC breakpoints with a warning against the use of these agents without additional therapeutic measures.Similarly, CLSI does not publish susceptible MIC breakpoints for colistin against any Gram-negative pathogen.
Imipenem/relebactam inhibited over 99% of NME and 100% of the putative AmpC/ESBL-positive non-CRE phenotype E. coli and K. pneumoniae.Imipenem/relebactam was specifically developed to retain activity against isolates carrying KPCs and generally retains in vitro activity against those without MBL-type carbapenemases and has variable activity against organisms with OXA-48-like enzymes.Geographic differences in β-lactamase prevalence and other resistance mechanisms affect the in vitro activities of all currently available β-lactams and β-lactam/β-lactamase inhibitor combinations, including ceftolozane/tazobactam and imipenem/ relebactam.In the current study, we observed low numbers of MBLs, KPC, OXA-48-like and GES carbapenemases in both Enterobacterales and P. aeruginosa (Figure 1).Therefore, mechanisms of carbapenem resistance other than β-lactamases (e.g.OprD mutations in combination with AmpC hyperproduction in P. aeruginosa) likely predominated in the isolates we studied.
In conclusion, recent clinical isolates of Enterobacterales and P. aeruginosa collected in Israel from 2018 to 2022 showed high susceptibility (≥93%) to ceftolozane/tazobactam and imipenem/ relebactam.If the in vitro data described here translate into successful clinical results, ceftolozane/tazobactam and imipenem/ relebactam could be vital treatment options for patients in Israel with infections caused by Gram-negative pathogens, including antimicrobial-non-susceptible and -resistant Enterobacterales and P. aeruginosa.

c
Levofloxacin was only tested against Enterobacterales isolates from 2018 to 2021.d Putative ESBL non-CRE phenotype was defined by an isolate testing with a CAZ MIC ≥ 2 mg/L and a MEM MIC ≤ 1 mg/L.

Table 1 .
Antimicrobial susceptibility of clinical isolates of Enterobacterales and P. aeruginosa collected in Israel 2018-22 For IPM, CAZ, FEP, TZP and LVX against P. aeruginosa, the results represent '% susceptible, increased exposure' as defined in the EUCAST guidelines.